Figure 5.

Lack of endothelial PECAM-1 does not affect T-cell arrest or post-arrest T-cell crawling and diapedesis. Confluent WT and PECAM-1^−/− pMBMEC monolayers were stimulated with IL-1β (20 ng/ml) for 16–24 h prior to live cell imaging. Activated encephalitogenic CD4⁺ T cells were allowed to arrest on pMBMECs at low flow (0. 1 dyn/cm²). After 5 min, the flow was increased to 1.5 dyn/cm². For time-lapse videos, pictures were taken every 5 s for a total of 20 min. (A) The number of arrested T cells after increasing the flow rate (frame 60). (B) The behavior of each arrested T cell was determined for the subsequent 15 min (frame 60 to 240) and assigned to one of the following categories: detachment, probing behavior, continuous crawling, diapedesis. (C) For all T cells eventually starting to crawl, the time span between initial arrest to the pMBMECs and the first frame of crawling was determined. (D–F) Each T cell crawling on the activated endothelial monolayer was tracked and the crawling speed (D), accumulated crawling distance (E), and Euclidian crawling distance (F) were calculated. (G) For all crawling T cells eventually starting to transmigrate, the time span between halt of movement and start of diapedesis was calculated. (H) For all T cells that finished diapedesis, the duration of diapedesis was calculated. (I) T cells, which eventually started to transmigrate, were classified according to their prior behavior: crawling or stationary. Data are derived from 9 videos per pMBMEC genotype performed in three independent experiments. Error bar, ± SD; FOV, field of view; ns, not significant. Reported statistics result from unpaired, two-tailed Student's t-tests. Test statistics for (C): p = 0.0488, t(16) = 2.132 (d = 1.066); ^*p < 0.05; ns, not significant.